| Breast cancer is one of the most common malignant tumors in women in the world,and its incidence is increasing year by year.According to the American Cancer Society(ACS),the number of new cases of breast cancer in2023 will reach 300,000,still surpassing lung cancer as the world’s leading cancer.Therefore,from the molecular mechanism of the occurrence and development of breast cancer to the drug therapy to find molecular targets,breast cancer research is a hot spot However,with the deepening of breast cancer research,the targets of action other than the molecular classification of breast cancer are still being explored.In order to obtain better clinical benefits,it is a difficult and hot topic for medical researchers to find specific targets for diagnosis and treatment of breast cancer from the molecular mechanism.Centromere protein U(CENPU)is a protein coding gene,and the protein encoded by centromere protein is one of the important members of the centromere protein family.The genome of human CENPU gene was located on chromosome 4 4q35.1,which was composed of 2494 base pairs.The protein encoded by CENPU gene was composed of 418 amino acids with a molecular weight of 47.522k Da.Centromere is a special chromatin domain that exists throughout the cell cycle,and the brief assembly of centromere during mitosis occurs on this platform.CENPU is an important part of it.They are involved in the formation of CENPA-NAC(nucleosome associated)complexes,in which they act as scaffold proteins responsible for the initial recruitment and maintenance of centromeric PLK1 populations until their degradation.At the same time,CENPU recruits Plk1(Polo-like Kinase 1),one of the Polo-like kinases,and promotes its phosphorylation,which plays an important role in the correct localization of mitotic centromeres.Therefore,the complex formed with the participation of CENPU plays a central role in the assembly of centromeric proteins,mitotic processes,and chromosome segregation.With the continuous development of basic medicine,clinical medicine and basic medicine are closely linked,and the biological functions of CENPU are constantly discovered:CENPU is not only involved in cell mitosis,but also related to the regulation of cell cycle,oocyte maturation,angiogenesis and other signaling pathways.Recent studies have shown that CENPU is abnormally expressed in a variety of human malignant tumors.CENPU is involved in the biological process of cancer development and is associated with the prognosis of cancer patients.Further studies showed that CENPU,as a new biological marker,has the characteristics of promoting the proliferation,migration and invasion of cancer cells,specifically in promoting the formation of tumor blood vessels,promoting epithelial cell interstitium and so on.Therefore,CENPU has been gradually discovered as a potential biological target.However,due to the heterogeneity of the tumor,there are many differences in its role in different malignant tumors.In non-small cell lung cancer,for example,it promotes tumor progression through the Wnt/β-catenin signaling pathway and is associated with poor prognosis in patients.In primary hepatocellular carcinoma,Notch signaling pathway promotes the proliferation of hepatocellular carcinoma cells.In lung adenocarcinoma,CENPU promotes tumor progression through the PI3K/AKT signaling pathway.CENPU is being continuously explored in different malignancies and has been reported as a new biological target.However,the expression,mechanism and clinical significance of CENPU in invasive breast carcinoma need further study.This study was divided into three parts to study the expression and clinical significance of CENPU and its family in invasive breast cancer:Part One:The key genes of CENPU in triple negative breast cancer were screened by bioinformation analysis,and the expression of CENPU in breast cancer and its relationship with prognosis were verified by bioinformation analysis.Part Two:Through screening,key gene CENPU and its several family mem-bers play a core role in breast cancer,to determine the expression of breast cancer-related CENP protein family members in breast cancer and the correlation with prognosis,and to further reveal its interaction mechanism and signaling pathway.Part Three:Triple negative breast cancer samples were collected,and the expression of CENPU protein in 85 cases of primary breast cancer tissues was detected by immunohistochemical Max Vision TM one-step method.The expression level of CENPU protein was analyzed by expression intensity and expression ratio,combined with clinicpathological indicators of breast cancer patients and collected follow-up data.The correlation between CENPU and clinical indicators and prognosis was analyzed.Part One CENPU is highly expressed in triple negative breast cancer and is a key gene in the occurrence of breast cancerObjective:To screen the key genes of triple negative breast cancer by bioinformation method,and their expression and clinical significance in breast cancer.Methods:1.from the GEO(Gene Expression Omnibus)database(https://www.ncbi.nlm.nih.gov/geo/)to obtain the Gene Expression data set in this study.It contains two independent data sets GSE65194 and GSE38959.Data set GSE65194 was composed of 55 triple negative breast cancer tissue samples and 11 healthy breast tissue samples,and data set GSE38959 was composed of 30 triple negative breast cancer tissue samples and 13 normal breast tissue samples.Utilize online tooling GEO2R(http://www.ncbi.nlm.nih.gov/geo/geo2r)Screening out differential genes(DEGs)from two data sets(P<0.01,|log FC|≥2),And use Venn diagram to obtain the intersection of different genes in two data sets.Online STRING database(version11.0;https://string-db.org/)were used to construct PPI networks for these differential genes.Subsequently,Cytoscape(version 3.6.0)is an open source bioinformatics software platform that matches visual analysis of the PPI network and identifies key genes using Cytoscape Plug-in Molecular Complex Assay(MCODE).After further labeling of up-regulated and down-regulated genes,PPI networks were mapped using Cytoscape.The most important modules in the PPI network are also identified using MCODE.Meanwhile,we collected DEGs of 8 modules through MCODE APP,and 8 seeds of each module(with the highest MCODE score for each module)were screened as key genes.2.An online database DAVID database with integrated biological data and analysis tools(DAVID;http://david.ncifcrf.gov)(version 6.8)Conduct GO enrichment analysis on these differential genes to obtain enrichment information about these genes,including biological process(BP),cellular component(CC),and molecular function(MF).KEGG(The Kyoto Encyclo-pedia of Genes and Genomes)enrichment analysis obtained the enriched signal pathways of these differential genes.3.UCSC Xena Functional Genomics Explorer(https://xenabrowser.net/)was used to construct hierarchical cluster analysis of key genes to verify the expression of CENPU,a key gene of the most important module,in breast cancer tissue and normal breast tissue.4.Using online tools TIMER2.0(https://cistrome.shinyapps.io/timer/)visual CENPU in different subtypes expression in breast cancer and generic carcinoma tissue.At the same time with Oncomine databases and network resources based on the cancer omics data UALCAN database(http://ualcan.path.uab.edu/)were used for validation..5.Using survival analysis tools Kaplan Meier plotter10(http://kmplot.com/analysis/)analysis CENPU m RNA expression level with relapse-free survival(recurrence free survival,RFS)and overall survival(overall survival,OS),the relationship between the It is validated against the Progno Scan database.6.Based on Linked Omics database,the positive and negative correlation genes with CENPU in breast cancer were obtained,and these genes were enriched and analyzed.Using KEGG(https://www.genome.jp/kegg/pathway.html)signaling pathway chart,to get the related genes associated with MAPK signal pathway,GEPIA(http://gepia.cancer-pku.cn/)was used to analyze the relationship between CENPU and related genes in the signaling pathway.Results:1.Two triple negative breast cancer data sets GSE398959 and GSE65194were standardized and 795 and 2984 differential genes of breast cancer were obtained,respectively.The intersection of the two groups of differential genes yielded 438 differential genes.2.Build PPI network through STRING platform and visualization through Cytoscape software.A total of 269 up-regulated genes and 169 down-regulated genes were identified.According to MCODE algorithm of Cytos-cape,8 modules and 8 key genes(the highest scoring genes in each module)were obtained:CENPU,GZMB,HIST2H2BE,HMGCS2,ICAM1,SDC1 and SEMA6D.Among them,CENPU,GZMB,HIST2H2BE,ICAM1 and SDC1were up-regulated genes,while COL14A1,HMGCS2 and SEMA6D were down-regulated genes.Meanwhile,there were 102 up-regulated genes in the most important module,the key gene of which was CENPU.3.UCSC database visualized 8 genes in breast cancer tissue,and expression levels of all up-regulated and down-regulated key genes were consistent with the above conclusions,with P<0.05.4.GO enrichment and KEGG enrichment analysis.GO enrichment showed that the first three biological processes(BP)were enriched in cell division,mitotic nuclear division and sister chromatid condensation.The first three cell components(CC)of DEGs were enriched in concentrated chromo-some centromeres,chromosomes,centromeric regions and centromeres.The first 3 molecular functions(MF)were mainly concentrated in protein binding,microtubule binding and ATP binding.KEGG analysis showed that this module was mainly concentrated in the cell cycle,oocyte meiosis and oocyte maturation mediated by progesterone,and might be associated with tumor-related pathways such as p53 signaling pathway,Fanconi anemia pathway and Fox O signaling pathway.5.CENPU was highly expressed in most pancarcinomas,including breast cancer,lung adenocarcinoma,bladder cancer,stomach cancer,endometrial cancer,glioblastoma,lung squamous cell carcinoma,bile duct carcinoma,colon cancer,etc.The expression levels of all subtypes of breast cancer were increased(P=6.51E-53).6.UALCAN analysis on the expression level of CENPU in breast cancer results:CENPU was highly expressed in cancer tissues.Among the subtypes,the expression of the triple-negative subtype was the highest,followed by the HER-2 positive type,and then the Luminal subtype.The expression of CENPU was the highest in young breast cancer patients,and decreased with the increase of age.7.Kaplan-Meier survival analysis results:High CENPU expression was associated with poor recurrence-free survival(P<1e-16)and overall survival(P<2.6e-7).8.CENPU related genes were mainly concentrated in MAPK signaling pathway and other related pathways,and were correlated with genes in its signaling pathway.Conclusions:1.CENPU,as a key gene of triple negative breast cancer,has been identified in the database.It plays a core role in the most important module.From database genome chip large sample data,the results are reliable.From database genome chip large sample data,the results are reliable.2.Differential genes(DEGs)with CENPU as the key gene mainly play a role in cell mitosis,and play a functional role in chromosome and centromeric region.It is involved in cell cycle,oocyte meiosis,oocyte maturation media-ted by progesterone,p53 signaling pathway,Fanconi anemia pathway,Fox O signaling pathway,etc.3.CENPU is highly expressed in a variety of pancarcinomas,including breast cancer,suggesting that CENPU may be a potential biological marker of malignant tumors.4.Compared with normal breast tissue,the expression of CENPU in all subtypes of breast cancer is highly expressed,among which the expression level of triple-negative breast cancer is the highest,followed by HER-2positive type and Luminal subtype,suggesting that the expression of CENPU may be positively correlated with the malignancy of breast cancer.5.High CENPU expression is associated with poor recurrence-free survival(RFS)and overall survival(OS)of breast cancer.6.CENPU may be involved in promoting the occurrence and development of breast cancer through MAPK signaling pathway.Part two The expression and significance of CENP family in breast cancerObjective:To investigate the expression,interaction mechanism and prognostic significance of CENP family in breast cancerMethods:Coexpedia(http://www.coexpedia.org/)was used to analyze the coexpression network of CENPs in the Gene Expression Omnibus(GEO)database,get 10 genes of CENP family associated with breast cancer.Visua-lize the expression of 10 CENP family genes in pancancer using Oncome database(https://www.oncomine.org/).The differential expression of 10CENP family genes in breast cancer tissues and normal breast tissues was analyzed based on the UALCAN(http://ualcan.path.uab.edu)database.Kaplan-Meier Plotter(www.kmplot.com)database was used to further investi-gate the relationship between 10 CENP family genes and relapse-free survival(RFS)and overall survival(OS)in breast cancer patients.c Bio Portal further analyzed CENPs gene changes and the relationship between gene changes and survival outcomes in breast cancer patients.Then Breast Cancer Gene-Expression Miner(BC-genex-miner v4.5),Gene MANIA(http://genemania.org)and STRING analyze the mechanisms by which members of 10 protein families interact at the gene and protein levels.Finally,the Cluster Profiler package 4.0.2 of R software was used to analyze the GO enrichment and KEGG pathway enrichment of CENPU-related genes.Results:1.10 CENP genes related to breast cancer were screened from 19 CENP family genes,namely,CENPF,CENPE,CENPU,CENPA,CENPN,CENPI,CENPK,CENPW,CENPM and CENPL.2.10 CENPs m RNA was overexpressed in almost all cancer tissues,especially in breast cancer,lung cancer,colorectal cancer and sarcoma.In addition,all CENPs m RNAs,especially CENPF,CENPE,CENPU,CENPA,CENPN,CENPM,and CENPL genes,were overexpressed in breast cancer tissues compared with corresponding normal tissues.Among them,CENPF,CENPE,CENPU,CENPA and CENPM were the most reported CENPs in the breast cancer data set.The UALCAN database further confirmed that 10CENPs were overexpressed in all breast cancer samples compared to normal tissue samples(P<0.0001).3.Kaplan-meier survival curve showed that,except CENPK(HR=1.12,P=0.13),overexpression of CENPs was associated with poor RFS(P<0.01).In addition,the other nine CENPs had p values well below 0.01.In addition,poor OS was associated with high CENPF(HR=1.79,P=1.6E-09),CENPE(HR=1.44,P=0.00015),CENPU(HR=1.64,P=2.6E-07),CENPA(HR=1.57,P=2.8E-06),CENPN(HR=1.47,P=1.8E-05),CENPI(HR=1.25,P=0.022)and CENPM(HR=1.49,P=3.3E-05)expression.There was no significant difference in OS between patients with high and low expression of CENPK(HR=1.11,P=0.46),CENPW(HR=1.2,P=0.18)or CENPL(HR=1.15,P=0.31).In fact,for CENPK,the RFS difference between the two groups was statistically significant(P=0.0059)when patients were divided into two groups using the optimal cut-off value rather than the median expression value.4.Frequency of CENPs mutations in breast cancer:CENPF,16%;CENPE,5%;CENPU,4%;CENPA,4%;CENPN,10%;CENPI,4%;CENPK,4%;CENPW,5%;CENPM,2.9%;CENPL,14%.The main types of mutations include amplification,deep deletion,high m RNA,and missense mutations.There was no difference in survival between patients with and without CENP mutations,DFS(P=0.248)and OS(P=0.321).5.Pearson’s correlation analysis results showed that there was a signifi-cant positive correlation between each CENP pair(all p values were<0.0001),and complex interaction networks directly existed among CENP family mem-bers at both gene and protein levels.6.GO enrichment analysis showed that CENPs were mainly located in chromosomes,centromere region,kinetosomal region,condensed chromo-some and centromere region,and were mainly involved in the assembly of centromere complex,the assembly of CENPA-containing nucleosome,chro-matid tissue containing CENPA,and the assembly of nucleosome and assem-bly of nucleosomes unrelated to DNA replication,.And plays other roles in microtubule binding proteins and microtubule binding.KEGG enrichment analysis showed that these genes were involved in oocyte maturation,cell cycle and oocyte meiosis mediated by progesterone.Conclusions:1.In the CENP family,10 family genes CENPF,CENPE,CENPU,CENPA,CENPN,CENPI,CENPK,CENPW,CENPM,and CENPL have been identified,which are associated with breast cancer.2.These 10 CENP genes are highly expressed in breast cancer compared with other cancer species.CENPs are highly expressed in breast cancer compared with normal breast tissue and is associated with poor prognosis in breast cancer patients.3.10 CENP gene mutation types in breast cancer are mainly concen-trated in gene amplification,deep deletion,high m RNA and missense mutation,and the mutation is not correlated with disease-free survival and overall survival of breast cancer.4.10 CENPs were positively correlated at the gene level.CENPA is most closely related to CENPE and CENPW.They constitute a complex interactive network,which is manifested in the formation of complex,promoting the same signal path and so on.5.CENPs are mainly located in chromosomes,centromere region,kinetosomal region,condensed chromosomes and centromere region,and are mainly involved in the assembly of centromere complex,the assembly of CENPU-containing nucleosome,and other signaling pathways mediated by progesterone such as oocyte maturation,cell cycle and oocyte meiosis process.Part Three Expression of CENPU in triple negative breast cancer and its relationship with clinical parameters and prognosisObjective:To investigate whether CENPU is correlated with menopausal status,age of menarche,tumor size,lymph node metastasis status,clinical stage and survival prognosis of breast cancer patients,and to determine whether this biological target has potential clinical significance.Methods:From December 2013 to January 2015,85 patients with primary breast cancer admitted to the Breast Center of the Fourth Hospital of Hebei Medical University and confirmed by pathology with complete follow-up data were collected.Paraffin-embedded tissue specimens,clinicopatholo-gical data and follow-up data were collected.To analyze the relationship between the expression of CENPU in breast cancer tissues and the character-istics of clinical pathological indicators such as menopausal status,age of menarche,tumor size,clinical TNM stage,P53,KI-67,TOPOⅡ,etc.Kaplan-Meier method was used to calculate DFS and OS,and multivariate Cox proportional risk analysis was used to analyze risk factors affecting survival.Chi-square test was used for CENPU and clinical indicators.Results:1.Relationship between the expression of CENPU and clinicopathologic features of breast cancer1)CENPU expression status in primary breast cancer:Among 85 patients with primary breast cancer,the staining sites of CENPU were mainly located in the cytoplasm,and the positive signal showed a brown granular appearance(Fig.1,2,3,4),with negative expression in 9 cases(10.6%),positive expres-sion in 76 cases(89.4%),and h score of 6 or above in 21 cases(24.7%).2)The relationship between the expression of CENPU and the character-istics of clinical pathological indicators such as menopausal status,age of menar-che,tumor size,clinical TNM stage,number of lymph nodes,P53,KI-67,TOPOⅡ:Positive expression rate of CENPU at menopausal status(x~2=0.667,P=0.414),age at menarche(x~2=2.053,P=0.152),tumor size(x~2=0.004,P=0.951),clini-cal TNM staging(x~2=7.844,P=0.097),number of lymph nodes(x~2=0.013,P=0.314)、P53(x~2=1.930,P=0.165)、KI-67(x~2=1.351,P=0.245)、TOPOⅡ(x~2=1.338,P=0.247)and other clinical and pathological indicators were not significantly different between the groups.2.Relationship between CENPU expression and prognosis of breast cancer1)Relationship between CENPU expression and DFS and OS of breast cancerThe 85 patients were followed up for a median of 60 months,26 patients developed local recurrence or distant metastasis,and 23 patients have died.DFS at 72 months were 52.38%(11/21)in CENPU positive group and23.43%(15/64)in CENPU negative group.There were significant differences in DFS between the two groups(P=0.0038,Fig.5).The 72 months OS of CENPU positive group was 42.86%(9/21),and that of CENPU negative group was 21.88%(14/64).There were significant differences in OS between the two groups(P=0.037,Fig.6).As shown in the following figure(Fig.7,8),DFS(P=0.0011)and OS(P=0.0048)between CENPU groups were significantly different according to h scores(0,1,2,3,4,6,9).2)Multi-factor analysis of COX regression model affecting DFS and OSVariables included in COX multivariate analysis were tumor size,TNM pathological stage,P53,number of lymph node metastases,menopausal status,age of menarche,Ki-67 expression,TOPOII expression status,and h-score(CENPU).The results showed that age of initial menstruation,tumor size and lymph node metastasis were independent risk factors for DFS and OS in breast cancer patients,h score was an independent risk factor for DFS,tumor stage and Ki-67 expression were independent risk factors for OS in patients with breast cancer(P<0.05,Table.2).P53,menopausal status,Ki-67expression,TOPOII had no significant correlation with DFS and OS(P>0.05).Conclusions:1.The expression of CENPU protein in breast cancer is not correlated with menopausal status,age of menarche,tumor size,TNM pathological stage,and lymph node metastasis.2.The expression of CENPU protein in breast cancer is not related to P53,KI-67 and TOPOⅡ.3.The expression level of CENPU in breast cancer is negatively correlated with DFS and OS.4.Menstrual age,tumor size and lymph node metastasis are independent risk factors for DFS and OS in patients with breast cancer;CENPU is an independent risk factor for DFS;tumor stage and Ki-67 expression are independent risk factors for OS in patients with breast cancer. |
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